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      Glyco-Analytical Multispecific Proteolysis (Glyco-AMP): A Simple Method for Detailed and Quantitative Glycoproteomic Characterization

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          Abstract

          Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.

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          Author and article information

          Journal
          Journal of Proteome Research
          J. Proteome Res.
          American Chemical Society (ACS)
          1535-3893
          1535-3907
          September 09 2013
          October 04 2013
          September 20 2013
          October 04 2013
          : 12
          : 10
          : 4414-4423
          Affiliations
          [1 ]Asia Glycomics Reference Site, Daejeon 305-764, South Korea
          [2 ]Cancer Research Institute, Chungnam National University, Daejeon 305-764, South Korea
          [3 ]Department of Chemistry, University of California—Davis, Davis, California 95616, United States
          [4 ]Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, South Korea
          [5 ]Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang 363-883, South Korea
          Article
          10.1021/pr400442y
          24016182
          © 2013
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